Protocols for LCM preparation and analysis
For both I & II Frozen & Paraffin-EmbeddedTissue Sections
Staining should be performed as close as possible
to the scheduled LCM transfer time using solution baths that are replaced
regularly. Staining should be performed as follows:
For optimal transfer of frozen tissue sections it is best to keep sections < 10 µm thick. Thicker sections are more difficult to visualize. If there are folds in the tissue the cap may not make direct contact with the enitre surface at that area. Therefore it is advisable to inspect the tissue before placing down the cap. If any tissue seems to be mounded or folded, it is best not to place the cap over that area.
The tissue section must be dry and not coverslipped for effective LCM transfer. The staining appears darker and more granular due to light scattered from the irregular air-tissue interface. The tissue where the polymer melts and bonds after laser activation appears lighter and resembles a coverslipped slide due to the replacement of the air in the tissue with the polymer. This phenomenon is called index-matching or "polymer wetting."
Poor transfers may result if the slide is not
fully dehyrated (i.e. the 100% ethanol becomes hydrated after repeated
use). The final xylene rinse facilitates the efficiency of transfer with
LCM. If a tissue section does not transfer well, a longer xylene rinse
may help. While other staining protocols can be used, the slides should
be dehydrated in a final xylene step.
After microdissection (roughly 500-1,000 cells),
the cap is inserted into an Eppendorf tube containing digestion buffer
(50 ul buffer containing 0.04% Proteinase K*, 10 mM Tris-HCL (pH 8.0),
1 mM EDTA, and 1% Tween-20). The tube is placed in a 370C
oven to equilibriate. It is then placed upside down so that the digestion
buffer contacts the tissue on the cap. The incubation continues overnight
at 370C. The tube is centrifuged for 5 minutes and
the cap is removed. The reaction is heated to 950C
for 8 min to inactivate the proteinase K. It can then be used directly
as template for PCR (please see PCR section of RT-PCR below). We have used
this extraction method for the following methods: LOH analysis, dideoxy
fingerprinting (DDF), clonality analysis (chromosome X inactivation) and
direct sequencing of PCR products for single base mutational analysis.
*All of the concentrations in this protocol refer to the reagent concentrations, not the final concentration of the mixes.
The cap post laser transfer should be placed tightly onto the Eppendorf tube. The tube is then inverted back and forth over the course of 2 minutes. It is then quick spun to collect all of the buffer. The cap may be removed and replaced with another LCM cap or the investigator may proceed with the RNA extraction. Roughly 1,000-5,000 cells are extracted for most RNA applications. While not quantitative, the intensity of the pink color of the extraction buffer also enables the investigator to gain a sense of the amount of tissue dissected.
It is important to DNAase microdissected samples for applications where DNA would interfere. These include RT-PCR with primers that would amplify DNA and cDNA library construction involving an adapter ligation step. The DNAase used should be certified RNAase free. We have used this protocol to obtain RNA that was subsequently used in the following protocols: RT-PCR, cDNA library construction (with adapter ligation or homopolymer tailing methods), cDNA microarray probe and modified differential display. The RT-PCR protocol described below is for both random primed RT and oligodT (for enrichment of mRNA and 3' message analysis) RT. The (+) RT means that reverse transcriptase is added to the RT reaction. The (-) RT means that the reverse transcriptase is replaced by water. By running a (-) RT it is possible to discern whether the primers used for PCR could be amplifying DNA. There would be no cDNA in the (-) RT reaction. Therefore any amplified product must be due to DNA.
The PCR protocol presented here includes incorporation
of radioactivity into the PCR products. For highly abudant cDNAs or clear-cut
PCR products it may be possible to discern products on an ethidium bromide
treated agarose gel (replace P32 volume in protocol below with water).
However, for low abundant transcripts or when PCR product patterns are
complicated (i.e. polymorphic markers for LOH), visualization on acrylamide
with radioactive incorporation may be necessary. It is listed that 0.2
µL of 20uCi/µl P-32 per 10 µL PCR reaction is used. This
amount of radioactivity often results in visible products in less than
2 hrs exposure. It is possible to reduce the radioactivity to 0.05 µL
P32 per 10 µL PCR reaction. The exposure times will be increased.
Small pieces of tissue are often embedded in paraffin
wax prior to cutting and mounting onto glass slides. The advantages of
paraffin embedding over freezing tissue include improved histology and
convenient storage. Prior to embedding, however, tissue must be "fixed"
to preserve cellular morphology and prevent autolysis. While the routine
fixative formalin preserves the tissue morphology, it cross-links RNA and
DNA to protein limiting the analysis of nucleic acids. (Mies 1994) DNA
derived from microdissected tissues fixed in formalin are still amenable
to PCR amplification. The amplification products will be reduced in quantity
and size compared to frozen tissues. We routinely amplify 200 bp products
from formalin fixed paraffin embedded tissues using the DNA extraction
procedure outlined above. Alcohol based fixatives improve the integrity
and recovery of the nucleic acids (Foss 1994) and transfer well by LCM.
Once fixed, the tissue is embedded in paraffin. Paraffin processing can be performed via a routine overnight or accelerated cycle in an automated tissue processer. Once embedded, thin sections are mounted onto glass slides. Consistent LCM transfers have been demonstrated from 5-10 µm thick paraffin embedded tissue sections. For a successful LCM transfer, the strength of the bond between polymer film and the targeted tissue must be stronger than that between the tissue and the underlying glass slide. Therefore, for most tissue types, techniques which increase the adhesion of the tissue section to the glass slide should be avoided. These include the sectioning of tissue onto charged or positive glass slides, and the use of an adhesive in the water bath prior to tissue mounting.
To prevent cross-contamination while sectioning,
paraffin and tissue fragments should be wiped from the area with xylenes
between each slide. If possible, a fresh microtome blade should be used
for each block. The prepared tissue slides should be stored at room temperature
until required for LCM transfer.
Staining should be performed as close as possible to the scheduled
LCM transfer time using solution baths that are
replaced regularly.
RNA Isolation (modified Stratagene Microisolation protocol)
Re-extracton of RNA (same reagents as above)
Reverse Transcription (final volume 20 uL)
Components and cycling will depend on individual template and primers
Reaction components:
Sample Temps/Times for PCR in PE 9600 thermocycler
P.A.G.E. *(onlyif radioactivity is used)